利用双向电泳比较3种提取户尘螨蛋白的方法

Comparison of three extracting methods for dermatophagoides pteronyssinusproteins by Two-dimensional electrophoresis

为改进户尘螨过敏原提取方法 ,在采用Coca s液、裂解液及Trizol法提取纯种户尘螨螨体蛋白后 ,用二喹啉甲酸 (BCA)检测法测定蛋白质含量 ,双向电泳比较上述蛋白提取方法的效果。结果显示 ,在蛋白质回收率方面 ,裂解液优于Trizol法优于Coca s液。经过双向电泳 ,Coca s液提取的蛋白仅仅有少量低分子量的蛋白点 ;用裂解液提取的蛋白可见明显增多低分子量的蛋白点 ,但无中分子量的蛋白点 ;Trizol法提取的蛋白可见中分子量的蛋白点如174～ 178ku和 133 0ku的蛋白质。另外 ,纯种户尘螨螨体经Trizol液提取后进行双向电泳 ,在考染和银染时均出现 5个高丰度的特殊蛋白点 ,一个为酸性中分子量蛋白 ,位置孤立的 ;另有 4个位置彼此非常接近的中性蛋白点。比较后结论为使用Coca s液提取的尘螨蛋白质浓度低 ,蛋白点少。使用裂解液提取的尘螨蛋白质浓度虽然较高 ,但是没有中分子量的蛋白点 ,不能全面反映尘螨的过敏原。使用Trizol法提取的蛋白质浓度中等 ,有中分子量蛋白点 ,反映的蛋白点更加全面。纯种户尘螨螨体经Trizol液提取后进行双向电泳 ,在考染和银染时出现的特征性蛋白点 ,作为一种双向电泳的指纹图谱 ,在鉴定此种纯种户尘螨螨体时具有一定的价值。

To explore the extract methods of Dermatophagoides pteronyssinus (D. pteronyssinus), the extracts were prepared with Coca's solution, lysis solution of two-dimensional (2D) electrophoresis and Trizol reagent respectively. In protein concentration assay, BCA reagents were used. The protein concentrations in the extracts by different methods were: lysis solution >Trizol reagent >Coca's solution. Two-dimensional electrophoresis was done with these different extracts. The experiment results were: 1) many protein spots at low molecular weight (MW) in extract with Coca's solution method; 2) more protein spots at low MW in extract with lysis solution method; 3) several medium MW protein spots(e. g. 174-178ku ) and more low MW protein spots in extract with Trizol reagent. There are five peculiar proteins in every 2D map in our experiment (twelve times) with Trizol reagent. There were low protein concentration and less protein spots with Coca's extract. There were few protein spots at medium MW with lysis solution though they have higher protein concentration. It was shown that Trizol reagent extracted more protein components of D. pteronyssinus PMB than that by Coca's solution and lysis solution. The five peculiar proteins in 2D map with Trizol was reagent considered as one of finger printing of D. pteronyssinus PMB in 2D electrophoresis map.